A campaign targeting a conserved Hsp70 binding site uncovers how subcellular localization is linked to distinct biological activities.

The potential of small molecules to localize within subcellular compartments is rarely explored. To probe this question, we measured the localization of Hsp70 inhibitors using fluorescence microscopy. We found that even closely related analogs had dramatically different distributions, with some residing predominantly in the mitochondria and others in the ER. CRISPRi screens supported this idea, showing that different compounds had distinct chemogenetic interactions with Hsp70s of the ER (HSPA5/BiP) and mitochondria (HSPA9/mortalin) and their co-chaperones. Moreover, localization seemed to determine function, even for molecules with conserved binding sites. Compounds with distinct partitioning have distinct anti-proliferative activity in breast cancer cells compared with anti-viral activity in cellular models of Dengue virus replication, likely because different sets of Hsp70s are required in these processes. These findings highlight the contributions of subcellular partitioning and chemogenetic interactions to small molecule activity, features that are rarely explored during medicinal chemistry campaigns.